DESCRIPTION (Verbatim from the Applicant's Abstract): Combinatorial biology is a powerful approach for harnessing the tremendous biosynthetic capability of microorganisms, including primary and secondary pathways. A key aspect of this overall approach is the identification and characterization of genes and enzymes that prescribe the assembly of complex natural product molecules. Although access to rich genomic diversity is essential, tools for expression of genes in an appropriate microbial host is equally important to successful development of broad-based combinatorial biosynthetic systems. The aim of the proposed work is to develop a more thorough understanding of the functionality of individual polyketide synthase (PKS) modules (loading, processing, and termination), the mechanism of metabolic branching leading to construction of two distinct ring systems, the basis for flexible substrate specificity of two key tailoring enzymes, and overall pathway regulation in the pikromycin (pik) biosynthetic pathway from Streptomyces venezuelae. The pikromycin system includes a set of 18 genes residing on 60 kb of DNA with a locus containing two resistance genes, a polyketide synthase locus encoding six modules and a type II thioesterase, a desosamine biosynthetic locus comprised of 9 genes, a single cytochrome P450 hydroxylase and a putative gene involved in pathway regulation. In the first stage of this project, the role of a unique beta-ketosynthase domain will be investigated and a series of hybrid PKSs containing engineered loading domains will be constructed and analyzed for production of novel natural product modules. Second, the unusual ability of the pik PKS to generate 12- and 14-membered ring macrolactones will be studied to elucidate the genetic and biochemical basis for generating structural diversity. A series of engineered PKS systems will be assembled and based on contraction and expansion of the modular system in order to probe molecular recognition and metabolic flexibility. Finally, the mechanism of polyketide chain termination will be explored and hybrid PKSs constructed through manipulation of the thioesterase domain and thioesterase II of the pik PKS. Overall, we expect this work will provide key information on substrate specificity and function of the various pik-encoded catalytic domains and enzymes and provide insight into how PKS modules can be engineered to create novel biologically active molecules.